Quantitative Online Monitoring of an Immobilized Enzymatic Network by Ion Mobility–Mass Spectrometry (Open Access)

Abstract: The forward design of in vitro enzymatic reaction networks (ERNs) requires a detailed analysis of network kinetics and potentially hidden interactions between the substrates and enzymes. Although flow chemistry allows for a systematic exploration of how the networks adapt to continuously changing conditions, the analysis of the reaction products is often a bottleneck. Here, we report on the interface between a continuous stirred-tank reactor, in which an immobilized enzymatic network made of 12 enzymes is compartmentalized, and an ion mobility–mass spectrometer. Feeding uniformly 13C-labeled inputs to the enzymatic network generates all isotopically labeled reaction intermediates and products, which are individually detected by ion mobility–mass spectrometry (IMS–MS) based on their mass-to-charge ratios and inverse ion mobilities. The metabolic flux can be continuously and quantitatively monitored by diluting the ERN output with nonlabeled standards of known concentrations. The real-time quantitative data obtained by IMS–MS are then harnessed to train a model of network kinetics, which proves sufficiently predictive to control the ERN output after a single optimally designed experiment. The high resolution of the time-course data provided by this approach is an important stepping stone to design and control sizable and intricate ERNs.

Recommended citation: Quentin Duez, Jeroen van de Wiel, Bob van Sluijs, Souvik Ghosh, Mathieu G. Baltussen, Max T. G. M. Derks, Jana Roithová, Wilhelm T. S. Huck. (2024). " Quantitative Online Monitoring of an Immobilized Enzymatic Network by Ion Mobility–Mass Spectrometry." Journal of the American Chemical Society. 146(30), 20778–20787.
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